| Rheumatoid arthritis（RA）is a major chronic destructive disease worldwide,characterized by joint swelling,synovial inflammation and cartilage and bone destruction.Although the cause of pathogenesis and pathophysiology of RA is still unknown,studies have shown that the role of lymphocytes is crucial in the pathogenesis of RA.The immune organs of the body are mainly divided into two parts:central immune organs and peripheral immune organs.Central immune organs,including thymus and bone marrow,occur early in embryonic development;peripheral immune organs,including the spleen and lymph nodes,appear late in embryonic development.A wide range of immune cells,including T lymphocytes,B lymphocytes and dendritic cells.The PGE2 levels in serum of RA patients are significantly increased.It is noteworthy that PGE2 regulates intracellular levels of cyclic adenosine monophosphate（cAMP）through G protein-coupled prostaglandin E2 receptor（EP receptor）signaling pathways.G protein-coupled receptor kinases（GRKs）,which are important negative regulators of G protein-coupled receptors（GPCRs）,cause phosphorylation of GPCRs that promotes the binding of arrestins to GPCRs and desensitizes GPCRs.According to the results of the previous study,the expression of GRK2 was significantly changed in rats with adjuvant arthritis（AA）and collagen-induced arthritis（CIA）;however,GRK2is particularly effective for the EP signal in T lymphocytes.The regulation of EP4 signal has not been reported.The new active monomer drug-paeoniflorin-6-oxy-benzene sulfonate（code CP-25,patent number ZL201210030616.4）is a new type of active monomer obtained by esterification modification of paeoniflorin（Pae）in the early stage of the research group.,showing strong anti-inflammatory and immunomodulatory effects.Studies have shown that CP-25 can improve RA symptoms by modulating PGE2-EP4-cAMP and TNF-alpha-TNFR1-TRADD-TRAF2-NF-κB signaling pathways,and CP-25 can also be regulated by BAFF and TNF-alpha-TRAF2-NF-KappaB signaling pathway mediates classical and non-canonical NF-κB signaling pathways to moderately regulate B-cell function to exert anti-inflammatory and immune effects.At the same time,the study also found that CP-25 can improve overall indexes of AA rats and reduce proinflammatory cytokines.Secretion levels significantly improve RA symptoms.In this study,the expression of GRKs and EP receptor subtypes was studied in the AA rat model with the thymus and spleen tissues of AA rats as the main research object,and flow cytometry and immunofluorescence were used in spleen-derived T cells.ELISA and co-immunoprecipitation and other technologies have profoundly studied the changes of T cell function and the mechanism of action of CP-25 in AA rats under inflammatory conditions,and further revealed the anti-inflammatory and anti-inflammatory effects and characteristics of CP-25.Objective:Observing the expression of EP receptor subtypes and GRKs in thymus and spleen tissues of AA rats,and revealing the functional changes of spleen-derived T cells in the inflammatory state of AA rats,providing a theoretical basis for elucidating the role of CP-25 in regulating T cell function.Methods:The AA model was induced by complete Freund’s adjuvant and inflammation began to appear on the 17th day after the model was established.The experiments were grouped as follows:Normal group,AA model group,and CP-25（50 mg/kg）group（CP-25 group）,8 rats in each group.All dosing groups were administered at a dose（50mg/kg）given by the body weight of the rats.From the 17th day,they were administered orally once a day for 13 consecutive days.The normal group and the model group were given intragastric administration by parallel administration of the same solvent CMC-Na solution according to the rat body weight.The degree of paw swelling was measured every 3 days.The rats were fasted one day before sacrifice,and the femoral artery was bleed.The thymus and spleen tissues were separated.The weight of each group of rats’thymus and spleen was weighed,and the thymus index and spleen index of each group were calculated.Thymus and spleen tissues were taken and sections were made for pathological examination,including spleen HE staining,and immunohistochemical staining of thymus and spleen;The distribution and expression of GRK2,3,5,6 and EP1,2,3,4 in thymus and spleen tissues were observed by immunohistochemistry;GRK2,3,5,6 and EP1,2,3,4 were observed by Western Blot in each group;immunofluorescence laser confocal method was used to detect the co-expression of GRK2 and EP2 and GRK2 and EP4 in each group.In order to detect the changes of T cell function and the role of CP-25 in the inflammatory state of AA rats,we first stimulated T cells with ConA in vitro and then added CP-25(10-6 mol/L).The CCK8 method was used to detect splenic T cell proliferation.And the effect of CP-25,followed by the detection of inflammatory cytokines interleukin-2（IL-2）and interferon-γ（IFN-γ）levels secreted specifically by T cells by ELISA.Then using flow sorting technology,high purity T cells were obtained.The level of cAMP in T cells was detected by ELISA.Finally,the expression of GRK2in T cells of each group was detected by immunofluorescence laser confocal microscopy.Immunofluorescence laser confocal Methods Combined with flow cytometry,the expression level of EP4 in T cells of each group was detected.Results:1.Changes of overall indexes in AA rats and the effects of CP-25At the beginning of the 17th day of the onset of AA rats,it was obvious that the feet swelling degree of the rat foot was obviously higher than that of the normal group;The spleen tissue of rats with HE showed spleen model rats compared to the normal group,the white pulp and red pulpline fuzzy,obvious hyperplasia of white pulp,appeared in germinal center,red pulp hyperplasia,congestion increased,and the outer medullary plexus of splenic sinus congestion,granulocyte infiltration;The thymus and spleen of AA rats were obviously enlarged,and the thymus index and spleen index increased significantly.CP-25 could significantly reduce the swelling of the feet of AA rats,improve the boundary between white pulp and red pulp,reduce granulocyte infiltration,and significantly reduce the thymus and spleen index of AA rats.2.Expression of GRKs and EPs in the thymus and spleen tissues of AA rats and the effect of CP-25.GRKs and EPs in thymus and spleen tissues of AA rats were detected by immunohistochemistry.It was found that the expression of GRK2,EP2 and EP4increased significantly,and the protein was expressed only in the red pulp area and the cortical part of the thymus;The results of Western blot detection showed that the expression of GRK2,EP2 and EP4 in the thymus and spleen tissues of AA rats increased significantly;Q-PCR detection found that the genes of GRK2 and EP4 in the thymus and spleen tissues of AA rats changed significantly.CP-25 could significantly reduce the protein expression level of GRK2,EP2 and EP4 in the thymus and spleen of AA rats.3.Co-expression of GRK2 and EP2,GRK2 and EP4 in thymus and spleen of AA rats and the effect of CP-25Co-expression of GRK2 and EP2,GRK2 and EP4 in the thymus and spleen tissues of rats by co-immunoprecipitation.The results showed that GRK2 and EP4co-expression in spleen of AA rats increased significantly compared with that of normal rats,while CP-25 significantly reduced the co-expression of GRK2 and EP4 in AA spleen.Confocal laser scanning confocal microscope further confirmed this result;The co-expression of GRK2 and EP2 in the spleen of AA rats had a tendency to decrease compared with the normal group.The dose of CP-25 after administration was higher than that of the AA model group,but there was no difference between the three groups.In thymus tissue of AA rats,GRK2 and EP2 and GRK2 and EP4 co-expressed normal group,model group and CP-25 administration group had no difference between the three groups.4.The splenic T cell proliferation and the effect of CP-25Compared with the normal group,ConA significantly promoted T cell proliferation.Compared with ConA group,CP-25 can significantly inhibit T cell proliferation.5.The level of inflammatory cytokines secreted by T cells of AA rats and the effect of CP-25Compared with the normal group,the proportion of spleen-derived T cells in AA model group and the expression of GRK2 and EP4 in spleen-derived T cells were significantly increased.Compared with AA model group,CP-25 could significantly down-regulate the proportion of spleen-derived T cells and the expression of GRK2 and EP4 in spleen-derived T cells.The expression of EP4 in spleen-derived T cells was detected by flow cytometry and the results were consistent with the above.6.The Level of cAMP in T Cells of AA Rats and the Effect of CP-25ELISA assay spleen homogenate supernatant,the results showed that compared with the normal group,AA model group,IL-2 and IFN-γlevels were significantly increased,CP-25 administration significantly decreased T-cell secretion of IL-2 and IFN-γlevels.7.Expression of GRK2 and EP4 in spleen-derived T cells and effect of CP-25Normal group,AA model group and CP-25 group of splenic lymphocytes were sorted out of high purity T cells（purity of 95%）,The level of cAMP in T cells was detected by ELISA.The level of cAMP in AA model group was significantly lower than that in normal group.CP-25 significantly up-regulated cAMP level in model group.Conclusions:1.CP-25 can effectively reduce the degree of joint swelling in AA rats and improve the histopathology of spleen in AA rats.2.The abnormal increase of GRK2,EP2 and EP4 in thymus and spleen tissues of AA rats at the inflammation stage,while CP-25 can significantly down-regulate the expression of GRK2,EP2 and EP4.3.The expression of GRK2 and EP4 in the spleen of AA rats increased,but CP-25 could significantly down-regulate their coexpression.4.The levels of IL-2 and IFN-γsecreted by T cells of AA rats were significantly increased,while CP-25 could significantly down-regulate their secretion.5.Abnormality of T cell function may be related to the EP4-cAMP signaling pathway,and CP-25 can moderately regulate T cell function by acting on the EP4-cAMP signaling pathway and exert anti-inflammatory and immune effects. |
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